1. Technical Field
The subject invention relates to methods of observing protein crystals so as to distinguish such crystals from other materials within a test sample as well as to obtain a vivid and precise image of the protein crystalline material of interest.
2. Background Information
In scientific studies of the three-dimensional atomic structure of biological macromolecules, X-ray or other diffraction experiments are extensively and widely used. These methods require the regular array of replicate molecules presented by highly ordered crystalline states of matter. Often great effort is expended in finding or screening conditions suitable for forming, growing, and harvesting crystals of sufficient size and quality. Further, the structure and mode of binding of a ligand to its target are often derived from the data.
In the area of drug discovery research, many ligand structures are desired in order to optimize and guide iterative medicinal chemistry synthesis toward achieving the binding properties desired for a drug molecule. For pharmacological reasons, the target of many drugs is a protein molecule so a large fraction of X-ray crystallography research in drug discovery is performed on crystals of proteins (see Anderson et al., Chem. Biol. 10(9):787-97 (2003)). Interest and effort are increasing in the attempt to obtain structures of membrane-bound proteins such as cell receptors.
Lacking direct molecular control to build a crystal at the molecular level, scientists rely on large numbers of tests to find conditions where a concentration or other gradient, such as those induced by vapor diffusion, will allow or drive a crystal to form. Such a crystal, or at least a zone within the crystal, must be solid, large enough, and relatively free of defects for it to yield good X-ray diffraction data. If a particular crystal growth test is successful, the location, size or form of the crystal is unpredictable (see McPherson et al., Structure 3:759-68 (1995)). Multiple crystals can form as well causing additional difficulties. Steps of adding crystal seeds or presenting crystal-inducing surfaces have been used but are not universally applicable.
Unfortunately, in the solutions and solution mixtures used to promote the formation or growth of a crystalline phase of a target macromolecule, very often other undesired solid material can form whose composition is unknown and can obscure or distract in the identification of crystals actually worthy of X-ray diffraction experiments (see McPherson, A., Preparation and Analysis of Protein Crystals, 1982, Krieger Publishing Co., Inc., Malabar, Fla., pp. 179-180). Salt, detergent, polyethylene glycol, lipids or other excipients can also form crystals (or precipitate). Some of these can have similar morphology or outwardly resemble the desired crystals. Amorphous precipitates, liquid phase separations or skins on droplets are also occasionally observed. These may be composed of, or include, protein to some degree. Therefore, with such a plethora of possibilities it becomes important to be able to monitor and identify protein crystals in such crystallization attempts, usually performed in multiple sites or chambers.
At the present time, 96-chamber plastic plates have gained popularity as a sample format for screening large numbers of crystallization trials. Using these plates, in the vapor diffusion method of crystallization, a protein solution is confined as a sitting droplet by a well. Crystals are relatively small and can form at various locations within the well. A basin below contains the reservoir liquid that slowly adjusts through vapor diffusion the protein solution droplet's concentration in crystallizing agents. Another well-known format used extensively in the past is to hang a droplet from a cover over the reservoir.
Existing methods of microscopy of materials have limitations in their application to these types of samples. Phase, birefringence, retardance, crossed-polarizer or other contrast methods using visible light and, for example, exploiting the difference in index of refraction between protein crystal and solution, may not be conclusive enough alone to allow convenient or rapid scoring of crystallization attempts. Cross polarization for example, uses the anisotropic nature of crystalline materials to refract light and produce birefringence. Birefringent crystals appear as rainbow colored objects against a dark background. Crystals with little structural anisotropy may not be birefringent, for example, the bacterial cell division protein FtsZ (Löwe, J. et al., Nature 391(6663): 203-6 (1998)). If the isotropic nature of protein crystals that grow from a given sample is not known before screening, the use of birefringence will result in some missed hits. Many organic and inorganic materials present in crystallization screens can also form birefringent crystals that result in false positives.
Absorbance or transmitted light microscopy in the UV for this purpose is difficult in most crystal growth formats. For spectral information, crystals are generally removed and mounted in instruments for examination (Bourgeois, D. et al., J. Appl. Cryst. 35:319 (2002)).
Chemical modification of a protein prior to crystallization (such as attaching a fluorescent probe, see Sumida, J. et al., J. Cryst. Growth 232:308-316 (2001)) in order to more easily visualize its crystals when they form is usually undesirable for the risk of denaturing the protein, or altering its biochemical, e.g. compound-binding, properties in subtle or major ways. The crystallization behavior of the protein may also be unpredictably altered.
In order to recognize protein crystals, dyes can be added to a crystallization well after crystals form that adsorb into or stain protein specifically [www.hamptonresearch.com; http://www.hamptonresearch.com/hrproducts/4710.html]; however, such a process is time consuming and invasive, as it can modify crystals substantially and can alter or abrogate the binding of any drug-like compound under study, and thus is limited to cases where the crystals need not be harvested.
In view of the above, a definite need exists for non-invasive methods that allow one to inspect crystals such that one can distinguish them from other materials in a sample as well as to visualize the crystals precisely.
All patents and publications referred to herein are hereby incorporated in their entirety by reference.